[Use of bacteriology in the campaign against caries].
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Biomedical subjects
Publications and source records attributed to J Slots.
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Since recent studies have implicated Actinobacillus actinomycetemcomitans in the etiology of localized juvenile periodontitis, this investigation determined the effectiveness of subgingival debridement, topical Betadine Solution, and systemic tetracycline in suppressing subgingival A. actinomycetemcomitans and other microorganisms. A total of 20 deep periodontal pockets and 10 normal periodontal sites of 6 localized juvenile periodontitis patients was included in the study. Each patient was treated in 3 stages over a period of 22 weeks, and the result of treatment was monitored for an additional 38 weeks. The first stage of treatment included plaque control, as well as thorough scaling and root planing, composed of at least 6 h of debridement. No concomitant periodontal surgery was performed. In the second stage, Betadine saturated cotton gauze was inserted into the periodontal pockets for 10 min. Stage 3 involved systemic tetracycline therapy (1 g/day) for 14 days. The subgingival microflora was determined at frequent intervals by selective culturing of A. actinomycetemcomitans and Capnocytophaga and by direct microscopic examination. The clinical effect was assessed by measuring changes in probing periodontal attachment level, probing periodontal pocket depth, radiographic alveolar bone mass, and other relevant clinical parameters. Scaling and root planing reduced the total subgingival bacterial counts and the proportions of certain Gram-negative bacteria, but no periodontal pocket became free of A. actinomycetemcomitans. Betadine application had little or no effect on the subgingival microflora. In contrast, tetracycline administered via the systemic route suppressed A. actinomycetemcomitans, Capnocytophaga, and spirochetes to low or undetectable levels in all test periodontal pockets. A. actinomycetemcomitans reappeared in 9 of the deep periodontal pockets after the administration of tetracycline. Most of these 9 pockets became free of detectable A. actinomycetemcomitans during the second week of tetracycline administration, whereas pockets which yielded no A. actinomycetemcomitans after tetracycline therapy became free of the organisms during the first week of tetracycline treatment. This data suggests that systemic tetracycline therapy of localized juvenile periodontitis should, as a practical rule, be continued for 3 weeks. Periodontal destruction continued in 4 deep pockets which all showed high posttetracycline A. actinomycetemcomitans counts. All 6 pockets which demonstrated a marked gain in periodontal attachment yielded no cultivable A. actinomycetemcomitans. No association was found between periodontal disease status and subgingival Capnocytophaga, spirochetes or motile rods. The present study indicates that A. actinomycetemcomitans is an important etiologic agent in localized juvenile periodontitis.(ABSTRACT TRUNCATED AT 400 WORDS)
This study was undertaken to evaluate the microbiological and clinical effects of a subgingivally applied mixture of H2O2-NaCl and NaHCO3 followed by subgingival irrigation with 1% Betadine in the treatment of periodontal disease. 20 adults with moderate to severe periodontal disease were included in a split mouth design study. All patients were given oral hygiene instruction and were subjected to supragingival scaling in all 4 quadrants, and subgingival scaling and root planing of half the dentition. 10 patients were instructed to use the chemical antimicrobial mixture twice a day instead of dentifrice, and also received professional application of the mixture once every 14 days for 3 months in connection with reinstruction in oral hygiene procedures. The remaining 10 patients received oral hygiene instructions combined with professional tooth cleaning without use of chemicals once every 14 days during a 3-month period. The effect of treatment was evaluated by monitoring the subgingival microflora, clinical periodontal parameters, and by computer assisted subtraction analysis of serial standardized radiographs to determine changes in mass of the supporting alveolar bone. The present study revealed that subgingival debridement combined with mechanical plaque control resulted in decreased numbers of subgingival microorganisms including spirochetes and motile rods, and arrested the progressive breakdown of the periodontal tissues. Topical antimicrobial agents used in combination with subgingival scaling further reduced the subgingival microflora and substantially improved early periodontal healing including gain of probing attachment level and gain in radiographic alveolar bone mass during the 12 months of observation. No clinical improvement but a tendency to further periodontal breakdown was found in the unscaled quadrants, even in those which were subjected to a personal application of the topical antimicrobial mixture. This study indicates that professional and personal subgingival application of a mixture of H2O2-NaCl and NaHCo3 will significantly enhance the microbiological and clinical effects of periodontal scaling and root planing. These agents, and the topical mode of antimicrobial therapy seem promising in the management of human periodontal diseases.
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The minimal inhibitory concentrations of a series of antimicrobial agents for human oral organisms were determined under anaerobic growth conditions by an agar dilution assay. With the exception of black-pigmented Bacteroides spp., minimal inhibitory concentrations for oral isolates were similar to those for non-oral isolates of organisms of the same or closely related species.
The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease.
Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.
On the basis of in vitro susceptibility testing of antibiotics, dyes, and other antimicrobial agents, we developed and evaluated a medium, TBBP, for the selective isolation of oral Capnocytophaga spp. TBBP medium consists of 4% Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.), 5% sheep blood, 0.1% yeast extract, 50 micrograms of bacitracin per ml, and 100 micrograms of polymyxin B per ml. A total of 34 Capnocytophaga stock cultures grew well on TBBP medium. Except for some streptococcal strains, TBBP medium inhibited growth of all test stock culture isolates of common oral gram-positive and gram-negative species. In a clinical study of 15 deep periodontal pockets, TBBP medium demonstrated Capnocytophaga recoverability that was similar to or higher than that shown by a nonselective blood agar medium. Typical Capnocytophaga colonial morphology enabled us to readily distinguish this organism from the few other bacteria which could grow on TBBP medium.
DNA was purified from 16 strains of Fusobacterium nucleatum and from five strains representing other Fusobacterium species. The relationships among fusobacteria were examined by DNA-DNA hybridization and by determining the guanine plus cytosine content of the DNA. F. nucleatum was found to comprise a heterogenous group of organisms related to Fusobacterium periodonticum and Fusobacterium simiae, but unrelated to any of the other species of Fusobacterium tested.
Isolates of Fusobacterium that differ from type strains of various fusobacterial species with respect to DNA sequence, cellular fatty acid composition, and biochemical activity, were obtained from periodontitis lesions in a patient with insulin-dependent diabetes mellitus. These isolates have the following distinguishing characteristics: 28% guanine + cytosine content; 40% or less DNA homology with type strains of representative fusobacterial species; cell size, 0.5 - 1 X 4 -100 microns; absence of motility; ability to ferment glucose, fructose, and galactose, but not 25 other carbohydrates; ability to produce indole; ability to hydrolyze hippurate but not esculin; sensitivity to bile; ability to produce little or no gas; ability to utilize threonine but not lactate. We propose that the organisms be classified as a distinct species of Fusobacterium to be named Fusobacterium periodonticum. The type strain of this new species has been deposited with the American Type Culture Collection under the designation ATCC 33693.
We compared the cellular fatty acid and protein content of 43 strains of oral fusobacteria isolated from patients with chronic gingivitis, acute necrotizing ulcerative gingivitis, and juvenile and adult periodontitis, as well as from the stump-tailed macaque, to that of eight reference strains of oral and non-oral Fusobacterium species. A gas-liquid chromatographic examination of trimethylsilyl derivatives of the fatty acid methyl esters revealed n14:0, 3-OH-14:0, n16:0, 16:1, 3-OH-16:0, n18:0, and 18:1 in each of the 51 study strains, and a variable occurrence of 14 other fatty acids. The n16:0 to 3-OH-16:0 ratio distinguished between Fusobacterium nucleatum and the non-oral species Fusobacterium varium, Fusobacterium necrophorum, Fusobacterium russii, Fusobacterium necrogenes, Fusobacterium mortiferum, and Fusobacterium naviforme. The soluble protein content, as determined by polyacrylamide gel electrophoresis, varied considerably among the oral fusobacterial strains studied, and underscored the heterogenous nature of these organisms. "Fingerprinting" of oral fusobacteria may be readily accomplished by polypeptide analysis.
Actinobacillus actinomycetemcomitans is a Gram-negative oral bacterium which has been implicated in the etiology of localized juvenile periodontitis. In this study, 403 subjects from four study groups were examined for A actinomycetemcomitans in subgingival dental plaque. Samples pooled from at least six periodontal sites were included from each subject. A actinomycetemcomitans was detected in 28 of 29 localized juvenile periodontitis patients but in only 15% of the other subjects including 28 of 134 adult periodontitis patients, 24 of 142 periodontally healthy subjects and 5 of 98 insulin dependent juvenile diabetics with varying degrees of gingivitis. A actinomycetemcomitans isolates from members of five families with localized juvenile periodontitis patients were biotyped on the basis of variable fermentation of dextrin, maltose, mannitol and xylose and serotyped by indirect immunofluorescence using serotype specific rabbit antisera. Individuals within a family all harbored A actinomycetemcomitans of the same biotype and serotype. However, even in families with individuals heavily infected with A actinomycetemcomitans, some family members did not appear to be infected with the organism. The apparent poor transmissibility of A actinomycetemcomitans between individuals may, in part, explain the overall low prevalence of localized juvenile periodontitis and the familial pattern of the disease. The high prevalence of A actinomycetemcomitans in the subgingival plaque of localized juvenile periodontitis patients, compared to the much lower prevalence in other patient groups, supports the hypothesis that A actinomycetemcomitans is an etiologic agent in this periodontal disease.(ABSTRACT TRUNCATED AT 250 WORDS)
The occurrence of subgingival Actinobacillus actinomycetemcomitans and Capnocytophaga in 12 localized juvenile periodontitis and 10 gingivitis patients from Panama was determined using selective culture techniques. A actinomycetemcomitans was present in all localized juvenile periodontitis lesions studied and was, on average, recovered in hundred-fold-higher numbers from localized juvenile periodontitis lesions than from gingivitis lesions. Capnocytophaga was only recovered in approximately threefold-higher numbers from localized juvenile periodontitis than from gingivitis. The study confirms and extends previous data indicating a close relationship between A actinomycetemcomitans and localized juvenile periodontitis. It is proposed that identification of A actinomycetemcomitans may be a valuable adjunct in the diagnosis of localized juvenile periodontitis.
Actinobacillus actinomycetemcomitans is an oral bacterium which is being encountered with increasing frequency in infective endocarditis. This organism occurs in high numbers in periodontitis lesions of patients with localized juvenile periodontitis (periodontosis). It is present infrequently, and only in low numbers in most other individuals. Its common resistance to penicillin, erythromycin and vancomycin represents a clinical problem in patients at risk of developing endocarditis after dental treatment. However, the high activity of tetracyclines against A. actinomycetemcomitans may be useful in prophylactic endocarditis considerations by allowing a suppression of the organism prior to the institution of recommended prophylactic protocols. In this study, we determined the effect of systemic tetracycline-HCl therapy (1 gm/day) on the oral A. actinomycetemcomitans population in five localized juvenile periodontitis patients who were heavily infected with the organism. A. actinomycetemcomitans could not be detected in samples of subgingival and supragingival dental plaque and cheek mucosal surfaces following 14 days of administration of systemic tetracycline. The organism was still undetectable 3 weeks after therapy but it reappeared at a few oral sites at week 8 post-treatment. On the basis of this data, it is proposed that the prophylactic endocarditis therapy of patients with high numbers of penicillin-resistant A. actinomycetemcomitans include a two-stage approach: first, the systemic administration of tetracycline for 14 days, and second, institution of a conventional prophylactic protocol during the time of dental treatment.
Serum antibodies to indigenous bacteria in ten beagle dogs were examined over a 7-month period during the development of ligature-induced periodontitis. Gram-negative strains comprised approximately 75% of the cultivable periodontitis microflora with a predominance of black-pigmented Bacteroides species. A total of 44 bacterial strains representing the predominant cultivable subgingival beagle dog microflora was selected for antibody determination. The IgG and, in some cases, IgM serum antibody titers to these organisms were determined by indirect immunofluorescence. The antibody titers to most test strains remained unchanged during the experimental period. Gram-negative bacteria generally exhibited lower titers than the Gram-positive bacteria. Especially low titers were found for the black-pigmented Bacteroides. Four dogs that developed the most severe periodontitis showed about 2-fold higher IgG titers to some Gram-negative anaerobic rods in the pre-ligation period than dogs that developed a more moderate periodontitis. These data suggested a possible diagnostic value of such antibody determinations. However, the overall finding of the present study was that serum antibody titers to key periodontopathic organisms remained low throughout the experiment. This result may suggest that the rapid periodontal destruction in ligature-induced periodontitis is due in part to an inadequate antibody response against the infecting microorganisms and their pathogenic products.
These studies demonstrate a unique constellation of organisms populating the subgingival area in periodontitis lesions of patients with juvenile or insulin-dependent diabetes mellitus (IDDM). The cultivable microflora was predominated by Capnocytophaga and anaerobic vibrios in the patients studied. In some patients, Actinobacillus actinomycetemcomitans were also found. This distinguishes the subgingival flora of IDDM patients suffering from periodontitis from that of patients with localized juvenile periodontitis (LJP), and that of adult periodontitis patients. In LJP most patients harbor both A actinomycetemcomitans and Capnocytophaga subgingivally; and in periodontitis lesions from nondiabetic adults, black-pigmented Bacteroides such as B gingivalis or B melaninogenicus subspecies intermedius are often found. Antibiotic susceptibility patterns suggest that penicillin or tetracycline or its analogs such as minocycline may be effective against the predominant cultivable microflora in periodontal lesions of IDDM patients; however, individual patients may harbor flora with significant resistance to these antibiotics.
IgG AND IgM ANTIBODY TITERS to eight bacterial isolates were measured by indirect immunofluorescence and ELISA in sera from acute necrotizing ulcerative gingivitis (ANUG) patients during the acute phase, from ANUG patients during the convalescent phase, from patients with gingivitis and from subjects with normal gingiva. Subjects were matched with respect to age and sex. Compared to the gingivitis and healthy groups, the ANUG groups exhibited significantly higher IgG and IgM titers to intermediate-sized spirochetes and higher IgG titers to Bacteroides melaninogenicus subsp intermedius. The findings support recent studies showing that these organisms are major bacterial components in ANUG lesions. They also suggest that these bacteria proliferate above-normal levels several weeks or months prior to the clinical onset of ANUG.